The polymerase chain reaction (PCR) is a powerful method for the rapid amplification of target nucleic acid sequences. PCR has facilitated the development of gene characterisation, including gene expression and/or regulation, and molecular cloning technologies including the direct sequencing of PCR amplified DNA, the determination of allelic variation, and the detection of infectious and genetic disease disorders. PCR is performed by repeated cycles of heat denaturation of a DNA template containing the target sequence, annealing of opposing primers to the complementary DNA strands, and extension of the annealed primers with a DNA polymerase. Multiple PCR cycles result in the amplification of the nucleotide sequence delineated by the flanking amplification primers. The incorporation of a thermostable DNA polymerase into the PCR protocol obviates the need for repeated enzyme additions and permits elevated annealing and primer extension temperatures which enhance the specificity of primer:template associations. Thermostable polymerases, such as Taq DNA polymerase, thus serve to increase the specificity and simplicity of PCR.
In many PCR based amplifications, a signal producing system is employed, e.g. to detect the production of amplified product. One type of signal producing system that is used in PCR based reactions is the fluorescence resonance energy transfer (FRET) system, in which a nucleic acid detector includes fluorescence donor and acceptor groups. FRET label systems include a number of advantages over other labelling systems, including the ability to perform homogeneous assays in which a separation step of bound vs. unbound labelled nucleic acid detector is not required. A primary problem with many prior art techniques is linked to the synthesis of dual labelled fluorescent oligonucleotides. European Patent Application EP1726664 discloses a detection system which overcomes this problem by using single-labelled oligonucleotide sequences of differing melting temperature (Tm) that hybridise to one another in free solution to form a fluorescent quenched pair (fluor/quencher cassette), that upon introduction of a complementary sequence to one of the sequences generates a measurable signal, one of the sequences being of a Tm that is below the annealing temperature (Ta) of the PCR process. In this system one of the single-labelled oligonucleotide sequences is preferably more than 10 bases longer than the other and more preferably at least 15 bases longer.
In detection systems using a labelled nucleic acid detector, high fidelity amplification is critical. Due to the nature of the PCR process and Taq DNA polymerase such methods can suffer from alternative side-reactions to the desired polymerisation reaction. For example, PCR can suffer from non-specific amplification when the reaction is assembled at ambient temperature. Taq polymerase retains a fraction of its activity at all temperatures and can therefore extend primers that are not complementarily annealed, leading to the formation of undesired products. The newly-synthesised region then acts as a template for further primer extension and synthesis of undesired amplification products. However, if the reaction is heated to temperatures of around 50° C. or above before polymerisation begins, the stringency of primer annealing is increased, and synthesis of undesired PCR products is avoided or reduced.
Primer-dimer is also a common side-reaction affecting PCR. Accumulation of primer-dimer occurs because of the hybridisation and extension of the primers to each other. Formation of primer-dimer results in the depletion of the reagents and hence overall reduction of PCR efficiency and/or the production of false positive results.
Hot-start PCR is a method to reduce non-specific amplification and hence limit the formation of non-specific PCR products including primer-dimers. Many different approaches have been developed to achieve this; see, for example, Moretti, T. et al. Enhancement of PCR amplification yield and specificity using AmpliTaq Gold DNA polymerase. Bio Techniques 25, 716-22 (1998) and Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance Nucleic Acids Res (2008) 36(20): e131. Such methods reduce the extension of primers following non-specific hybridisation prior to the start of PCR. However, such techniques only achieve partial alleviation of such problems since mis-priming events including primer-dimer formation can occur, although to a lesser extent, during PCR amplification. The use of PCR probes to detect the presence of a sequence internal to the PCR primers helps prevent the detection of any such non-specific products but adds significant cost to the process since a dedicated probe is required for each individual sequence to be detected. Cost effective high throughput genetic analysis requires the use of a universal detection system but in principle this can be impacted by the detection of non-specific amplification products.
There is a need for easy-to-synthesise, low cost and reliable, specific detection systems for use in the detection of primer extension products, e.g. in homogeneous PCR assays, which address the problems encountered with existing detection systems for PCR. The term homogeneous PCR assay is well known in the art, and is one where it is not necessary physically to separate the reaction components away from each other in order to derive the result of the reaction. The present invention is based on the finding that selection of the relative lengths of labelled oligonucleotide sequences that hybridise to one another to form a fluorescent quenched pair results in improvements in nucleic acid detection assay systems, particularly when used in a real-time setting. In the invention, the Tm of the fluor/quencher cassette is designed to be above the Ta of the amplification such that any unincorporated fluorescent oligonucleotide is hybridised to the quencher oligonucleotide at the fluorescence acquisition temperature allowing the reaction to be monitored in real-time or at end point. By adjusting the length and Tm of the quencher oligonucleotide it would be expected that the increased stability of the fluor/quencher cassette would simply inhibit PCR. However it is unexpectedly found that the specificity of amplification from the fluorescent primer is improved as shown by significant increases in the difference in Cq values (also known as Ct values) between samples and no template controls in real-time, or reduced detection of no template controls in end point applications.